The objective of this research program is to identify and examine determinants and additional factors that influence the overall phosphorylation state of the PhoQ histidine kinase. The primary way by which prokaryotes sense and respond to their environment is through the use of two component systems. The PhoP-PhoQ system is specifically required for virulence and/or growth in limiting Mg2+ in numerous gram negative bacteria. Understanding how the various intrinsic enzymatic activities and additional molecular factors combine to modulate the overall levels of PhoQ phosphorylation (and hence signal output of the PhoP-PhoQ system) remains an important unresolved problem in this field of study. One of the major foci of this program is to take advantage of our recent structural advances to elucidate and dissect the contributions of individual amino acids in the catalytic and nucleotide-binding properties of the PhoQ protein. We will use site-directed mutagenesis coupled with in vivo assays to determine the types and importance of various protein-nucleotide interactions in the autokinase reaction. These in vivo results will be compared to our previous in vitro enzymatic and binding studies to give a more complete picture of the autokinase reaction mechanism. The other major focus will be to investigate how acetyl CoA and the CpxA-CpxR regulatory pair (via perhaps a phosphohistidine phosphatase) input and integrate into PhoP-PhoQ signaling. More specifically we will isolate and characterize PhoQ mutants that are no longer repressed by extra cellular acetate (and intra-celllular acetyl CoA). We will also ascertain whether physiologically relevant cross-talk occurs between CpxR and PhoQ or whether a CpxR-regulated gene acts to dephosphorylate phospho-PhoQ. Due to their central role in bacterial physiology and virulence, and specifically PhoQ's role in the virulence of numerous gram negative pathogens, two-component systems are attractive antimicrobial targets. These experiments will undoubtedly prove useful in the isolation of antimicrobial agents.